Thyroglobulin (Tg) is the major protein of the thyroid, providing a matrix for synthesis of the thyroid hormones and a vehicle for their subsequent storage. It thus has a central role in thyroid hormone formation and metabolism. The long range objectives of this proposal are to define how Tg participates in the synthesis and intrathyroidal metabolism of hormones, and how variations in Tg structure and processing are involved in thyroid diseases, including familial goiter, nodular goiter, thyroid cancer, endemic goiter, and autoimmune thyroid disease. The specific aims are to: 1. Describe hormonogenic sits of Tg - This is done with radioiodine-labelled Tg from experimental animals by isolation, reduction, alkylation, and trypsin digestion of Tg followed by purification and sequencing of hormone containing peptides by HPLC and locating them by correlation with published sequences from cDNA clones of the Tg gene. In addition to describing the major sites, direct attention will be given to the sites whose utilization increased by TSH, and to the location of disulfides. Iodination in vitro of low iodine human Tg, followed by tryptic peptide processing as described above, will be used to identify early sites of iodotyrosyl formation and to study iodination cleavage of the Tg polypeptide chain. 2. Study Tg proteolysis - This project will continue the chemical characterization of the newly described thiol Tg hydrolase and will assess its role, and those of cathepsins D and B isolated lysosomes in the proteolysis of Tg to release thyroid hormones. Methods include: purification by column chromatography, enzyme characterization by affinity labelling, amino acid analysis and N-terminal sequencing; measurement of (125I) Tg digestion and identification of proteolytic products by gel electrophoreses and HPLC, comparing TSH-treated animals with controls. 3. Relate human Tg to thyroid disease - Tg will be isolated form diseased human thyroids obtained at surgery or autopsy, and tryptic peptides mapped on HPLC to identify variations in protein structure, with particular attention to hormonogenic sites.